The ability to monitor migration of biological cells through tight layer of other cells and tissues is crucial for understanding of mechanism of many life threatening diseases and development of modern therapeutic drugs. This migration is typically triggered by the presence of a particular chemical either immobilized on a surface or diffused through a tissue.
In inflammatory conditions, for example, the migration of leukocytes from blood vessels into diseased tissues is crucial to the initiation of normal disease-fighting inflammatory responses. At the same time, this process, known as leukocyte recruitment, is also involved in the onset and progression of debilitating and life-threatening inflammatory and autoimmunne diseases. Thus, the ability to control the migration of leukocyte through blood vessels into healthy tissues is an important pathway for development of therapeutic treatment. This migration is complicated by the fact that several leukocyte classes participate in this pathology (including lymphocytes, monocytes, neutrophils, eosinophils and mast cells) and each class carries out its own physiological function. There is the whole range of chronic autoimmune diseases. These include psoriasis, atherosclerosis, rheumatoid arthritis, contact dermatitis, multiple sclerosis, inflammatory bowel disease, hepatitis, sarcoidosis, idiopathic pulmonary fibrosis, dermatomyositis and diabetes. There are also numerous organ transplant rejection conditions such as allograft rejection and graft-versus-host disease that are determined to a large extent by the leukocyte migration.
The process by which leukocytes leave the bloodstream and accumulate at inflammatory sites and initiate the disease takes place in three distinct steps (Lawrence and Springer, 1991, Cell 65:859-73; Butcher E. C. 1991, Cell 67: 1033-36; Springer, T. A. 1990, Nature, 346: 425-33;). It is mediated by chemoattractant receptors, by cell-surface proteins, called adhesion molecules, and by the ligands that bind to these two classes of cell-surface receptors. The major types of adhesion molecules are known as “selectins”, “integrins” and “immunoglobulin (Ig) family” receptors.
Each of the three steps is essential for the migration of the leukocytes to target tissues. Blocking these steps has been shown to prevent a normal inflammatory response, and promotes abnormal responses of inflammatory and autoimmune diseases (Harlan et al., 1992, In vivo models of leukocyte adherence to endothelium. In Adhesion: Its Role in Inflammatory Disease., J. M. Harlan and D. Y. Liu, (eds.), W. H. Freeman & Co., pp. 117-150).
The three steps of leukocyte adhesion and transendothelial migration can be summarized as follows:
Step 1—Primary adhesion. Leukocytes attach loosely to the blood vessel endothelium and “roll” slowly along the blood vessel wall, pushed by the flow of blood. Leukocyte-endothelium attachment is mediated by cell surface adhesion molecules called “selectins” which bind to carbohydrate-rich ligands (“glycoconjugates”) on the leukocyte cell surface.Step 2—Activation of leukocytes and migration to the target site. Chemoattractant receptors on the surface of the leukocytes bind chemoattractants secreted by cells at the site of damage or infection. Receptor binding activates the immune defenses of the leukocytes, and activates the adhesiveness of the adhesion molecules that mediate Step 3.Step 3—Attachment and transendothelial migration. The leukocytes bind very tightly to the endothelial wall of the blood vessel and move to the junction between endothelial cells, where they begin to squeeze between these cells to reach the target tissue. This tighter binding is mediated by binding to adhesion receptors called “integrins” on the leukocytes to complementary receptors of the “Ig family” on the endothelium. (The Ig family molecules are named for their similarity to antibody molecules (immunoglobulins)). Chemoattractant receptors are also involved at this stage, as the leukocytes migrate up a concentration gradient of the chemoattractant secreted by cells at the target site.
It is increasingly clear that there may be another step preceding primary adhesion, i.e. preceding step 1, called “margination”. As a result of margination leukocytes get pushed by the red blood cells to the periphery of blood vessel, thereby allowing leukocytes to interact with the endothelium. However, margination is not commonly accepted as yet in the three step migration process described above.
These three steps of receptor-ligand interactions are all required and appear to act in a highly cooperative and coordinated manner to mediate leukocyte adherence to the microvasculature, diapedesis, and subsequent leukocyte mediated injury to tissue in inflammatory disease.
LFA-1 and Mac-1 together comprise the leukocyte integrins, a subfamily of integrins that share a common beta subunit (CD18) and have distinct alphaL, alpha M and alpha.X (CD11a, b and c) alpha subunits (Springer, 1990, Nature 346:425-433). They are required for leukocyte emigration as demonstrated by an absence of neutrophil extravasation (1) in patients with mutations in the common beta subunit (leukocyte adhesion deficiency), and (2) after treatment of healthy neutrophils with a monoclonal antibody (mAb) to the common beta subunit in vivo or in vitro.
The integrins LFA (lymphocyte function-associated antigen)-1 and Mac-1 on the neutrophil bind to the Ig family member ICAM (intercellular adhesion molecule)-1 on endothelium (Diamond et al., 1990, J. Cell Biol. 111:3129-3139). LFA-1 binds to ICAM-2, an endothelial cell molecule that is more closely related to ICAM-1 than these molecules are to other Ig superfamily members (Staunton et al., 1989, Nature 339:61-64).
The integrin VLA-4, that contains the alpha.4 (CD49d) subunit noncovalently associated with the beta1 (CD29) subunit, is expressed by lymphocytes, monocytes, and neural crest-derived cells, and can interact with vascular cell adhesion molecule-1 (VCAM-1) (Elices et al., 1990, Cell 60:577). Like ICAM-1 and ICAM-2, VCAM-1 is a member of the Ig superfamily (Osborn et al., 1989, Cell 59:1203).
Chemoattractants are soluble mediators which activate cell adhesion and motility and direct cell migration through formation of a chemical gradient. They are produced by bacteria and numerous cell types including stimulated endothelial and stromal cells, platelets, tumor cells, cultured cell lines, and leukocytes themselves. The cells responding to chemoattractants appear to express specific receptors on their surfaces which bind the chemoattractant molecules and sense the gradient. Receptor stimulation induces cells to respond via a common signal transduction pathway which involves interaction of the chemoattractant-receptor complex with a guanine nucleotide or GTP-binding protein (G protein). This interaction stimulates phosphatidyl inositol hydrolysis by a phospholipase C, thus generating inositol phosphates and diacylglycerol. A transient rise in cytosolic free calcium then activates protein kinase C, and a variety of events including protein phosphorylation, membrane potential changes, and intracellular pH alterations ensue.
Several of the chemoattractants primarily affecting neutrophils were among the first chemoattractants identified. These include the complement component C5a, arachidonate derivative leukotriene B4 (LTB4), platelet activating factor (PAF), and formylmethionyl peptides of bacterial origin such as formyl-met-leu-phe (fMLP). Although structurally dissimilar and stimulatory via separate receptors, these molecules produce a rapid and marked increase in neutrophil adhesiveness and motility leading to chemotaxis and prominent neutrophil accumulation in vivo. The receptors for C5a and fMLP have been identified and sequenced; cDNA clones for each have also been generated (Gerard and Gerard, 1991, Nature 349:614-617). These receptors share many structural features with one another and members of the “rhodopsin superfamily” of protein receptors.
The chemoattractants which predominantly activate and guide monocytes include monocyte chemoattractant protein-1 (MCP-1) (Leonard and Yoshimura, 1990, Immunol. Today 11:97-101), the RANTES protein (Schall et al., 1990, Nature 347:669-71), and the neutrophil .alpha. granule protein CAP37, among others. MCP-1 and RANTES are structurally homologous and belong to the subfamily of chemoattractive cytokines that are defined by a configuration of four cysteine residues in which the first two are adjacent (C—C). CAP37's structure is most homologous to proteins of the serine protease family (Peteira et al., 1990, J. Clin. Invest. 85:1468-76).
Compared with neutrophil and monocyte chemoattractants, little is known about chemoattractants for lymphocytes. The best characterized lymphocyte chemoattractants are RANTES and IL-8, which primarily attract monocytes and neutrophils. Several in vitro studies have described lymphocyte chemotactic activities in the culture supernatants of mixed lymphocyte reactions and mitogen-stimulated human peripheral blood mononuclear cells (Center and Cruikshank, 1982, J. Immunol. 128:2563-68; Van Epps et al., 1983, J. Immunol. 131:687).
Although considerable effort has been invested on the study of lymphocyte chemoattractants, they remain poorly characterized relative to monocyte and neutrophil chemoattractants. Chemoattractants for the latter cell types, such as MCP-1 and IL-8, have been purified based on the conventional chemotaxis assay, sequenced, and cloned. However, no molecule identified primarily as a lymphocyte chemoattractive factor has been sequenced and cloned.
The devices for studies and monitoring of transmigration of cells are well known in the fields of cell biology, life science, medicine, pharmaceutical and the area of drug development. There are also devices for filtering, growth and grouping of cells in these fields.
The most widely used assay is the Boyden Chamber assay, in which a microporous membrane divides two chambers, the lower containing the test chemoattractant and the upper containing the cells, e.g. lymphocytes. The microporous membrane is commonly nitrocellulose or polycarbonate, and may be coated with a protein such as collagen. The distance of migration into the filter, the number of cells crossing the filter that remain adherent to the undersurface, or the number of cells that accumulate in the lower chamber may be counted.
Such a Boyden chamber is also known as a transwell chamber. The chamber is made of well divided into two compartments, the upper and lower chamber, by a filter containing pores. A chemoattractant or other solution is placed in the lower chamber and the suspension cell is placed in the upper chamber. Cells can then migrate through the pores, across the thickness of the filter, and toward the source of chemoattractant. Cells that migrated across the filter and attached to the underside are then counted. In some assays the membrane is coated with Extra-Cellular Matrix (ECM) proteins (e.g. laminin, collagen, fibronectin) and then with endothelial cells (EC). A variety of devices of this class as well as the method for the transendothelial assay are described in U.S. Pat. No. 5,514,555 (Springer).
Boyden chambers are commonly used for studies of disease and also for the development of drugs for disease treatment. Here we list some examples of these applications:                Prostate cancer: It is not fully understood at present time the mechanism of the bone metastasis. However, interaction between cancer cells and bone environment (extra cellular matrix: ECM) seems critical for the process [Chen, N., et al., A Secreted Isoform of ErbB3 Promotes Osteonectin Expression in Bone and Enhances the Invasiveness of Prostate Cancer Cells. Cancer Res, 2007. 67(14): p. 6544-8]. The ability of prostate cancer cells to penetrate a synthetic basement membrane was assessed in a Matrigel-Boyden chamber invasion assay (BD Biosciences).        Allergy inflammation: the typical study is based on the eosinophils migration. Assay performed in a 48-well microchamber (neuroprobe) [Wong, C. K., P. F. Cheung, and C. W. Lam, Leptin-mediated cytokine release and migration of eosinophils: Implications for immunopathophysiology of allergic inflammation. Eur J Immunol, 2007].        Migration of vascular smooth muscle cells: key step in diseased arteries and may be controlled by ECM [Koyama, N., et al., Heparan sulfate proteoglycans mediate a potent inhibitory signal for migration of vascular smooth muscle cells. Circ Res. 1998. 83(3): p. 305-13].        Chemotactic ability of dental plaque: Whole plaque suspensions were chemotactic for polymorphonuclear leukocytes [Miller, R. L., L. E. Folke, and C. R. Umana, Chemotactic ability of dental plaque upon autologous or heterologous human polymorphonuclear leukocytes. J Periodontol, 1975. 46(7): p. 409-14].        
Boyden chambers/Transwell chamber and closely related devices are available from a number of vendors such as BD Biosciences; Corning; Neuroprobe; Millipore. Despite this, Boyden Chamber assays are typically associated with certain shortcomings. These include:                Intravital microscopy studies have suggested that leukocytes transmigration occurs over a time frame of minutes. In contrast, the readouts of most Boyden chamber transfilter assays are taken 1-4 hours after cell introduction. This excessive time span is necessary in order to get reasonable statistics of cell migration.        No physiological flow can be established in this assay thus is not possible to monitor cells though all stages of leukocyte recruitment.        There is no control of the gradient: chemokine diffusion in the body might be different than in vitro as it takes longer to get a cell migration on in vitro assays.        Changes in cell morphology during chemotaxis cannot be observed in real-time (because cells transmigrate through the filter).        
In addition, Boyden chamber assays cannot readily answer many questions related to the leukocyte migration. This is particularly true for the molecular and cellular mechanism of the chemokine-induced transendothelial migration step of leukocytes that still have not been fully elucidated. It is not clear at all if positive, negative or any chemokine gradients are involved and how such gradients may physically persist in relation to the endothelium. Initially it was thought that chemokines form soluble gradients across the blood vessel Endothelial Cells (ECs). However, in blood even a short persistence of a soluble chemokine gradient is not feasible because the constant flow of plasma removes the soluble chemokines from the site of their production. Therefore, it has been suggested already over a decade ago that only those chemokines that have been physically retained (immobilized) on the luminal membrane, for example by the glycosaminoglycan (GAG) residues of glycoproteins, may be able to effectively induce the integrin activation of the rolling leukocytes [Rot, A., Contribution of Duffy antigen to chemokine function. Cytokine Growth Factor Rev, 2005. 16(6): p. 687-94]. It is known though that a gradient of chemokine can direct cells and it is also well established that ECs protein receptors are necessary for cell adhesion.
There are other known assay types of assays for studies of cell migration. For example, the Dunn chamber assay comprises concentric rings separated by a bridge. The inner ring is filled with medium and the outer ring is filled with chemoattractant solution. Cells are cultured on a coverslip and placed upside down onto the Dunn chamber. The assays allow observation of migrating of cells towards the gradient formed between both rings.
Another area of applications that is broadly related to the area of transmigration is the growth of mammalian cells. A number of methods for culturing mammalian cells of different tissue origins have been reported. However, many of these cells are difficult to grow in vitro and, when grown, are not morphologically similar to in vivo tissue. It would be desirable to produce a tissue and cells which are morphologically similar to their in vivo counterpart for in vitro toxicology and other studies (for example, transepidermal drug transport).
Similarly there are requirements for tests of cells under continuous flow conditions resulting from the area of toxicity. Once identified, candidate drugs or modulators are usually evaluated for bioavailability and toxicological effects (Lu, Basic Toxicology, Fundamentals, Target Organs, and Risk Assessment, Hemisphere Publishing Corp., Washington (1985); U.S. Pat. No. 5,196,313 to Culbreth and U.S. Pat. No. 5,567,592 to Benet). Traditionally, early stages of drug discovery and development have concentrated on optimizing binding and potency of experimental compounds. Typically, animal studies are performed on late stage pre-clinical drug candidates to characterize pharmacokinetics (PK), pharmacodynamics (PD) and physiological toxicity. However, animal studies are costly, time-consuming and are limited, by throughput, to characterize no more than a few compounds. Furthermore, several drugs have shown unanticipated or unpredicted side effects only after reaching clinical trials or wide-scale release to the public. The pharmaceutical industry has the ultimate goal of replacing animal studies with in vitro tests that are validated, predictive models for human toxicity and drug dynamics. More recently, the industry has set a medium-term goal of creating high-throughput, in vitro tests that annotate candidate compounds with adsorption, metabolism and toxic (hereinafter referred to as “ADMET”) predictive parameters.
The toxicology of a candidate modulator can be established by determining in vitro toxicity towards a cell line, such as a mammalian, including human, cell lines. Candidate modulators can be treated with, for example, tissue extracts, such as preparations of liver (such as microsomal preparations) to determine increased or decreased toxicological properties of the chemical after being metabolized by a whole organism. The results of these types of studies are often predictive of toxicological properties of chemicals in animals, such as mammals, including humans. Current methods designed to model drug absorption in vivo involve growing a confluent layer of cells on a porous matrix that allows the test compound to permeate through the cell layer and matrix to a bottom well. It is desirable to carry out many of these measurements under conditions of continuous flow. These would mimic better the real physiological conditions. The complexity and interplay of biological processes that must be simulated to predict the ADMET properties of a compound far exceed the capabilities of currently available methods and tools. For example, when a patient takes a drug, it must first pass through the gastrointestinal tract and penetrate into the bloodstream. The drug must then survive oxidative modifications in the liver and get to the desired site (e.g., target organ or primary tumor) in a sufficient therapeutic concentration. Even if these biological functions could be faithfully reproduced in vitro, a difficulty remains in getting the capacity and format of the assay to facilitate testing and analysis of thousands of compounds. Ideally, the assays should be versatile enough to not only measure the enzyme cascade activity inside any living or whole cell, no matter what its origin might be, including cancer cells, tumor cells, immune cells, brain cells, cells of the endocrine system, cells or cell lines from different organ systems, biopsy samples etc., but should also be able to detect and measure the permeability of the cell to the candidate compound, as well as the metabolic activity of the cell on the candidate drug compound. Methodologies are desired that will allow for the more rapid acquisition of information about drug candidate interactions with enzymes that may potentially metabolize the candidate drug, earlier in the drug discovery process than presently feasible. This will allow for the earlier elimination of unsuitable compounds and chemical series from further development efforts, and also give an investigator insight as to the nature of metabolites with potential biological activity derived from the candidate drug. A parallel flow chamber may be used for this purpose. However, there are several disadvantages when using the parallel chamber. For example, the parallel flow chamber requires a substantial amount of the drug candidate for the experiment. Furthermore, setting up the experiment is often time consuming and rather complex.
By way of example, liver hepatocytes express a family of enzymes called cytochromes. One subfamily of cytochromes is known as cytochrome P450. The cytochrome P450 enzyme (CYP450) family comprises oxidase enzymes involved in the xenobiotic metabolism of hydrophobic drugs, carcinogens, and other potentially toxic compounds and metabolites circulating in blood. Efficient metabolism of a candidate drug by a CYP450 enzyme may lead to poor pharmacokinetic properties, while drug candidates that act as potent inhibitors of a CYP450 enzyme can cause undesirable drug-drug interactions when administered with another drug that interacts with the same CYP450. See, e.g., Peck, C. C. et al, Understanding Consequences of Concurrent Therapies, 269 JAMA 1550-52 (1993). Accordingly, early, reliable indication that a candidate drug interacts with (i.e., is absorbed by, metabolized by, or toxic to) hepatocytes expressing CYP450 may greatly shorten the discovery cycle of pharmaceutical research and development, and thus may reduce the time required to market a candidate drug. Consequently, such earlier-available, reliable pharmacokinetic information may result in greatly reduced drug development costs and/or increased profits from earlier market entrance. Furthermore, such earlier-available, reliable pharmacokinetic information may allow a candidate drug to reach the public sooner, at lower costs than otherwise feasible. Accordingly, extensive pharmacokinetic studies of drug interactions in humans have recently become an integral part of the pharmaceutical drug development and safety assessment process, e.g., Parkinson, A., 24 Toxicological Pathology 45-57 (1996).
Thus, despite the advances made to date, there remains a need to provide improved systems for carrying out cell based assays.